Bottom Line:
Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling.We develop antibodies that selectively target EphB4 and ephrinB2.Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development.

ABSTRACTBidirectional signalling is regarded as a notable hallmark of the Eph-ephrin signalling system: Eph-dependent forward signalling in Eph-expressing cells and ephrin-dependent reverse signalling in Ephrin-expressing cells. The notion of ephrin-dependent reverse signalling derives from genetic experiments utilizing mice carrying mutations in the intracellular region of ephrinBs. Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling. We develop antibodies that selectively target EphB4 and ephrinB2. We find that mice bearing genetically altered cytoplasmic region of ephrinB2 have significantly altered EphB4-dependent forward signalling. Selective inhibition of EphB4 using a functional blocking antibody results in defective lymphatic valve development. Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development.

Mentions:
The analogue-sensitive kinase allele (ASKA) technology is a powerful tool for determining the in vivo function of individual kinases3132. To exclude the possible off-target effect of NVP-BHG712, we took advantage of EphB4 ASKA (EphB4T693A/T693A) mice that are genetically engineered to carry a T693A mutation in the ATP-binding pocket, which sensitizes EphB4 to the inhibition by 1-Napthyl PP1 (NaPP1), an ATP-competitive inhibitor specifically designed to target mutant kinases. EphB4T693A/T693A mice have no apparent developmental or postnatal defects, indicating that the T693A mutation does not affect the normal function of EphB4. Similar to wild-type mice, EphB4T693A/T693A neonates displayed lymphatic valve defect after anti-ephrinB2 treatment, which was reversed when anti-EphB4 was co-administered (Fig. 7a). These results further confirmed that the T693A mutation did not affect the normal function of EphB4. Starting at P2, we treated the EphB4T693A/T693A neonates with daily dosing of NaPP1. The treatment caused a dramatic loss of valve structures in mesenteric lymphatic vessels (Fig. 7b). In contrast, NaPP1 had little impact on wild-type neonates (Fig. 7c), indicating that the effect of NaPP1 in EphB4 ASKA mice was due to specific inhibition of EphB4 activity. In addition to the loss of lymphatic valves, EphB4 inhibition also caused lymphatic vessel dilation, similar to anti-ephrinB2 treatment (Supplementary Fig. 8). As Napp1 inhibits the kinase activity of a targeted kinase, our findings suggested that the kinase activity of EphB4 is critical for regulating lymphatic valve morphogenesis. Although anti-EphB4 was able to reverse the effect of anti-ephrinB2, it failed to rescue the lymphatic valve defect resulting from NaPP1 treatment (Fig. 7b), indicating that the agonistic activity of anti-EphB4 depends on the kinase activity of EphB4. Interestingly, NaPP1 treatment of EphB4 ASKA mice had no impact on EphrinB2 phosphorylation, suggesting that NaPP1 is specifically targeting EphB4 forward signalling in EphB4 ASKA mice (Supplementary Fig. 9). Taken together, these studies using two independent approaches to inhibiting EphB4 provide strong evidence that EphB4, in particular its kinase activity, is required for lymphatic valve formation and maintenance in neonatal mice.

Mentions:
The analogue-sensitive kinase allele (ASKA) technology is a powerful tool for determining the in vivo function of individual kinases3132. To exclude the possible off-target effect of NVP-BHG712, we took advantage of EphB4 ASKA (EphB4T693A/T693A) mice that are genetically engineered to carry a T693A mutation in the ATP-binding pocket, which sensitizes EphB4 to the inhibition by 1-Napthyl PP1 (NaPP1), an ATP-competitive inhibitor specifically designed to target mutant kinases. EphB4T693A/T693A mice have no apparent developmental or postnatal defects, indicating that the T693A mutation does not affect the normal function of EphB4. Similar to wild-type mice, EphB4T693A/T693A neonates displayed lymphatic valve defect after anti-ephrinB2 treatment, which was reversed when anti-EphB4 was co-administered (Fig. 7a). These results further confirmed that the T693A mutation did not affect the normal function of EphB4. Starting at P2, we treated the EphB4T693A/T693A neonates with daily dosing of NaPP1. The treatment caused a dramatic loss of valve structures in mesenteric lymphatic vessels (Fig. 7b). In contrast, NaPP1 had little impact on wild-type neonates (Fig. 7c), indicating that the effect of NaPP1 in EphB4 ASKA mice was due to specific inhibition of EphB4 activity. In addition to the loss of lymphatic valves, EphB4 inhibition also caused lymphatic vessel dilation, similar to anti-ephrinB2 treatment (Supplementary Fig. 8). As Napp1 inhibits the kinase activity of a targeted kinase, our findings suggested that the kinase activity of EphB4 is critical for regulating lymphatic valve morphogenesis. Although anti-EphB4 was able to reverse the effect of anti-ephrinB2, it failed to rescue the lymphatic valve defect resulting from NaPP1 treatment (Fig. 7b), indicating that the agonistic activity of anti-EphB4 depends on the kinase activity of EphB4. Interestingly, NaPP1 treatment of EphB4 ASKA mice had no impact on EphrinB2 phosphorylation, suggesting that NaPP1 is specifically targeting EphB4 forward signalling in EphB4 ASKA mice (Supplementary Fig. 9). Taken together, these studies using two independent approaches to inhibiting EphB4 provide strong evidence that EphB4, in particular its kinase activity, is required for lymphatic valve formation and maintenance in neonatal mice.

Bottom Line:
Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling.We develop antibodies that selectively target EphB4 and ephrinB2.Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development.

ABSTRACTBidirectional signalling is regarded as a notable hallmark of the Eph-ephrin signalling system: Eph-dependent forward signalling in Eph-expressing cells and ephrin-dependent reverse signalling in Ephrin-expressing cells. The notion of ephrin-dependent reverse signalling derives from genetic experiments utilizing mice carrying mutations in the intracellular region of ephrinBs. Here we show that EphB4-dependent forward signalling regulates lymphatic valve development, a process previously thought to be regulated by ephrinB2-dependent reverse signalling. We develop antibodies that selectively target EphB4 and ephrinB2. We find that mice bearing genetically altered cytoplasmic region of ephrinB2 have significantly altered EphB4-dependent forward signalling. Selective inhibition of EphB4 using a functional blocking antibody results in defective lymphatic valve development. Furthermore, a chemical genetic approach is used to unequivocally show that the kinase activity of EphB4 is essential for lymphatic valve development.